The roles of peroxidase and aerobic dopa oxidase ("tyrosinase") in mammalian melanogenesis will be studied further by isolating and purifying these enzymes from transplantable mouse melanoma and cloned tissue cultures of mouse melanoma cells. These purified enzymes will be used to study their melanogenic potential (towards tyrosine and dopa), previously studied in histochemical preparation and in partially purified isolates. Currently, we have successfully cloned cultures of B16 and Harding Passy mouse melanoma for this purpose, and we have successfully obtained crude peroxidase and aerobic dopa oxidase for further purification. Enzymes will be purified by column chromatography, and purity of enzymes will be tested, using acrylamide gel electrophoresis. Function of enzymes will be assayed by spectrophotometric measurement of conversion of tyrosine and dopa to dopachrome, and by measuring conversion of labeled tyrosine to dopa. It will also be studied by measurement of tritiated water formed in the hydroxylation of tritiated tyrosine to tritiated dopa.